PROJECT SUMMARY/ABSTRACT Pancreatic cancer is almost always fatal and new approaches are needed to improve the prognosis for a disease that is now the third leading cause of cancer-related death. Pancreatic cancer cannot be cured without surgery, and unfortunately, nearly 90% of patients present with unresectable disease (locally advanced + metastatic), leaving patients and clinicians with very few treatment options once chemotherapy is completed. Radiation therapy cannot substitute for surgery because of morbid radiotoxicity to the nearby stomach and intestines that occurs before the tumor is controlled. Thus, treatment-related gastrointestinal (GI) radiation toxicity may be the single greatest barrier to improving treatment responses for unresectable pancreatic cancer. There are no known medications that can selectively protect the stomach and intestines from these side effects, but we previously published that the inhibiting signaling through EGLN proteins reduces radiation damage in a model of catastrophic radiation injury and now we propose to understand these effects in a clinically relevant system. Our laboratory's long-term goal is to develop therapies that reduce sequelae from radiation injury during clinically relevant and potentially curative cancer treatments. The central hypothesis is that inhibition of the EGLN enzymes, achieved through the use of the oral EGLN inhibitor FG-4592, will selectively protect the intestinal tract from radiation toxicity without protecting tumors. The objective of this grant is to uncover a deeper understanding of how the EGLN signaling axis modulates the radiation esponse in the intestinal stem cell niche and in pancreatic tumors in order to safely translate this technology to patients. The specific aims will test the following hypotheses: (Aim 1) EGLN inhibition reduces radiation toxicity to enable ablative stereotactic radiation for pancreatic cancer, which will improve survival; (Aim 2) EGLN inhibition works chiefly by stimulating the +4 intestinal stem cells, which will be tested with a lineage tracing experiment in reporter mice; (Aim 3) FG-4592 will selectively protect human intestinal tissue from radiation damage but not human pancreatic cancer. The proposed research is significant because FG-4592 has completed Phase III clinical trials for a non-oncologic indication and could thus be rapidly implemented as a radioprotector. This approach could be used potentially replace surgery with radiation for patients with unresectable pancreatic cancer and serve as the basis for a clinical trial in the next 5 years. This research is innovative because it takes a multidisciplinary approach to solving a complex clinical problem in an area with a significant unmet need. We use patient derived tumor organoids and intestinal ?mini-gut? cultures that have been generated at our institution to model this complex biology before a clinical trial with patients and moreover use cutting-edge techniques like single cell RNA seq to interrogate stem cell dynamics of the intestine in response to radiation injury and EGLN inhibition.